In this Mol Bio Minutes mini-episode of Speaking of Mol Bio, Dr. Andrea Hunger walks listeners through the practical differences between three core PCR approaches: endpoint PCR, quantitative PCR (qPCR), and digital PCR. Drawing on her experience in both academic research and industry, she explains how each technique provides different types of information and why choosing the right one depends on the biological question being asked.

Endpoint PCR is the simplest method and is ideal for basic presence-or-absence questions such as confirming cloning success or genotyping samples. While fast and accessible, it does not provide quantitative information. For experiments requiring measurement of gene expression levels or comparisons between samples, qPCR offers a powerful solution by monitoring amplification in real time and using Ct values and standard curves to estimate starting concentrations.

Hunger then discusses digital PCR, a newer technology that partitions samples into many micro-reactions to enable highly precise, absolute quantification of nucleic acids. Because it counts positive and negative partitions directly, digital PCR is especially valuable for detecting rare mutations, low-abundance targets, and applications like liquid biopsy analysis. Ultimately, she emphasizes that these PCR approaches are complementary tools, and the best experimental strategy is to choose the method that provides the level of information required for the next step in a research workflow.

Helpful resource links mentioned in this episode:

• Access educational eBook covering all three types of PCR and their use in gene expression analysis.
• Watch a video on when to choose digital vs. real-time PCR.
• Use the PCR primer design tool from Thermo Fisher.
• Access Harvard’s PrimerBank, a public resource of PCR primers.

Subscribe to get future episodes as they drop and if you like what you’re hearing we hope you’ll share a review or recommend the series to a colleague. 

Visit the Invitrogen School of Molecular Biology to access helpful molecular biology resources and educational content, and please share this resource with anyone you know working in molecular biology.

For Research Use Only. Not for use in diagnostic procedures.

In this Season 4 premiere of Speaking of Mol Bio, host Steve Lewis speaks with Dr. Nina Pollak from the University of the Sunshine Coast about the development of a single-dose vaccine aimed at protecting endangered koalas from Chlamydia pecorum. The disease is a major threat to koala populations across eastern Australia, contributing to infertility, blindness, and increased mortality. Pollak’s work focuses on optimizing the production and quality control of vaccine antigens while supporting regulatory approval and field trials that ultimately led to a conditional minor-use permit from Australia’s veterinary regulatory authority.

The vaccine relies on recombinant versions of the major outer membrane protein (MOMP) from three dominant chlamydial genotypes. Using standard molecular biology techniques, including gene cloning in E. coli, His-tag purification, SDS-PAGE verification, western blotting, and mass spectrometry, Pollak’s team produces and verifies these vaccine antigens before combining them with adjuvants to stimulate protective immune responses.

Beyond the science, the episode explores the challenges of conservation-focused vaccinology: field vaccination of wild animals, limited commercial incentives, and the importance of methods for monitoring disease prevalence. Pollak also reflects on the collaborative nature of the decade-long project and offers advice to young scientists pursuing difficult but meaningful research challenges.

Subscribe to get future episodes as they drop and if you like what you’re hearing we hope you’ll share a review or recommend the series to a colleague. 

Visit the Invitrogen School of Molecular Biology to access helpful molecular biology resources and educational content, and please share this resource with anyone you know working in molecular biology.

For Research Use Only. Not for use in diagnostic procedures.

In this Mol Bio Minutes mini-episode, Laurynas Alijošius shares how Rolling Circle Amplification (RCA) provides a reliable, high-yield approach for amplifying circular DNA prior to next-generation sequencing (NGS). This isothermal method avoids the need for thermal cycling and even bypasses the need for specific primers—making it ideal for challenging viral genomes, rare targets, or field samples.

Powered by the strand-displacing phi29 DNA polymerase, RCA amplifies DNA with impressive sensitivity and minimal GC bias. Laurynas breaks down the steps of multiple displacement amplification (MDA), explains why exonuclease-resistant primers are important, and explores how engineered polymerases like EquiPhi29™ DNA Polymerase dramatically improve yield and reduce reaction times. RCA products can be cleaned up and debranched to support a range of downstream workflows, including nanopore sequencing and transcriptomics.

From single-cell genomics to phage-based applications and in vitro expression systems, RCA is more than just a pre-NGS step; it’s a versatile tool with broad utility. Whether you're stabilizing viral RNA or tackling ultra-low-input samples, RCA and whole genome amplification offer new flexibility for today’s demanding sequencing workflows.

Subscribe to get future episodes as they drop and if you like what you’re hearing we hope you’ll share a review or recommend the series to a colleague. 

Visit the Invitrogen School of Molecular Biology to access helpful molecular biology resources and educational content, and please share this resource with anyone you know working in molecular biology.

For Research Use Only. Not for use in diagnostic procedures.